MOLECULAR-CLONING, SEQUENCING AND EXPRESSION OF THE CDNA OF THE MITOCHONDRIAL FORM OF PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM HUMAN LIVER

In human liver, phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.32) i s about equally distributed between cytosol and mitochondria in contra st with rat liver in which it is essentially a cytosolic enzyme. Recen tly, the isolation of the gene and cDNA of the human cytosolic enzyme has been reported [Ting, Burgess, Chamberlain, Keith, Falls and Meisle r (1993) Genomics 16, 698-706; Stoffel, Xiang, Espinosa, Cox, Le Beau and Bell (1993) Hum. Mel. Genet. 2, 1-4]. It was the goal of this inve stigation to isolate the cDNA of the human mitochondrial form of hepat ic PCK. A human liver cDNA library was screened with a rat cystolic PC K cDNA probe comprising sequences from exons 2 to 9. A cDNA clone was isolated which had overall a 68 % DNA sequence and a 70 % deduced amin o acid sequence identity with the human cytosolic PCK cDNA. Without th e flanking 270 bases (= 90 amino acids) each at the 5' and 3' end, the sequence identity was 73 %, on the DNA and 78 %, on the amino acid le vel. The isolated cDNA had an open reading frame of 1920 bp; it was 54 bp (equivalent to 18 amino acids) longer than that of human or rat cy tosolic PCK cDNA. The isolated cDNA was cloned into the eukaryotic exp ression vector pcDNAI and transfected into human embryonal kidney cell s HEK293; PCK activity was increased by 3-fold in the mitochondria, wh ich normally contain 70 % of total PCK activity, but not in the cytoso l. The isolated cDNA was also transfected into cultured rat hepatocyte s; again, PCK activity was enhanced by about 40-fold in the mitochondr ia, which normally possess only 10 % of total PCK activity, but not in the cytosol. In the rat hepatocytes only the endogenous cytosolic PCK and not the transfected mitochondrial PCK was induced 3-fold with glu cagon. Comparison of the amino acid sequences deduced from the isolate d cDNA with human and rat cytosolic PCK showed that the additional 18 amino acids were located at the N-terminus of the protein and probably constitute a mitochondrial targeting signal. Northern-blot analyses r evealed the human mitochondrial PCK mRNA tn hr 2.25 kb long, about 0.6 kb shorter than the mRNA of the cytosolic PCK. Primer extension exper iments showed that the 5'-untranslated region of mitochondrial PCK mRN A was 134 nucleotides in length.